Research with membrane proteins is uncovering physical rules that control the orientation and insertion of transmembrane segments into lipid bilayers. In general, the topology of a membrane protein is established primarily by the nature of the charged amino acids immediately adjacent to individual transmembrane segments. However, more research is required for polytopic membrane proteins to clarify the roles played by long-range structural interactions in the process of insertion of individual transmembrane segments. The proposed research introduces an experimental system that is particularly well-suited for study of polytopic membrane protein topogenesis. The approach uses fusion proteins between E. coli maltose binding protein (MBP) and the membrane protein of interest. The MBP reporter domain is attached to the N-terminal of the membrane protein and serves as a molecular probe for topology mapping or monitoring mutational alterations in the orientation of transmembrane segments. Topology and segment orientation are determined by proteolysis of the fusion protein. Digestion is limited to the membrane protein domain due to the resistance of MBP to proteolysis. The MBP fusion approach will be used to study effects of long-range interactions in membrane proteins on their assembly in the membrane. The tetracycline resistance protein (Tet) of the cloning vector pBR322 has been selected as a model membrane protein for development of the MBP fusion methodology. Recently, there has been a rapid increase in the number of human pathogens that are resistant to tetracyclines due to the spread of resistance genes via mobile genetic elements such as R-factors and transposons. The proposed research will provide detailed information about the structure of Tet proteins that will be useful in the design of Tet inhibitors.